Minimal interaction of support matrix and analytes is often desirable for separations such as gel electrophoresis and size exclusion chromatography of proteins. Proteins are well known to exhibit hydrophobic and/or ionic interactions with a variety of surfaces. Therefore, an inert material, which can significantly reduce or eliminate adsorption of proteins, would be very useful.
Known materials that resist protein adsorption include polysaccharide and polyacrylamide polymers; these enjoy wide application in gel electrophoresis and size exclusion separation of proteins1. An efficient method to address adsorption problems in capillary electrophoresis is to coat the capillary surface with such polymers2,3. In addition to polysaccharide and polyacrylamide, other neutral hydrophilic polymers have been investigated and found useful in capillary electrophoresis, such as polyvinyl alcohol4, polyethylene oxide5,6, polyvinylpyrrolidinone7 and a copolymer of polyethylene glycol and polypropylene glycol8. All of these polymers are neutral and hydrophilic. A systematic study of protein adsorption with a variety of surface structures resulted in the conclusion that materials are protein compatible if they are neutral, hydrophilic, proton acceptors and not proton donors9-11.
Other materials used in gel electrophoresis reported in 1992 by Zewert and Harrington are polyhydroxy methacrylate, polyhydroxy acrylate, polyethylene glycol methacrylate and polyethylene glycol acrylate12,13. To avoid the toxicities of acrylamide and bisacrylamide, and the difficulties associated with polyacrylamide gel electrophoresis of very hydrophobic proteins, such as bovine serum albumin or zein, polyethylene glycol methacrylate 200 in hydroorganic solvents was evaluated. Although there was no direct evidence to show the inertness of this material, successful electrophoresis of proteins demonstrated the protein compatibility of such polymers.
The inert polymers mentioned above are polymer gels that are soft in nature. These polymers can only be used in their swollen states because such polymers lose their permeabilities upon drying. Attempts have been made to prepare rigid beads with permanent porous structures from such polymers. Among these hydrophilic polymers, polyacrylamide is the only one that can form rigid beads by inverse suspension techniques using a high content of bisacrylamide as a crosslinker14. The use of a higher level of crosslinker accounts for the formation of rigid beads instead of soft particles.
Monolithic materials offer an alternative to columns packed with small particles or beads. A monolith (originally called a continuous bed or continuous polymer bed15) is a continuous rod with canal-like large through-pores and nanometer-sized pores in the skeletal structure. Preparation of a monolith is typically performed in a mold, such as in a tube or capillary where only one phase of the monomer mixture is used. Two types of monolithic materials have been developed to date. The first type is based on a silica backbone16,17 in which a continuous sol-gel network can be created by the gelation of a sol solution within a mold. Silica monoliths are mainly used for the separation of small molecules because of their hydrophobic characteristics after derivatization.
The second category includes polymer monoliths15,18 normally prepared by in-situ polymerization of monomer solutions, which are composed of a monomer, crosslinker, porogen and initiator. They can be initiated either by a redox system, e.g., TEMED and APS, or by a free radical initiator. For free radical initiation, both thermally and, more importantly, UV-initiated polymerization can be used. By the use of UV-initiated polymerization, a spatially defined monolith in a capillary or microchip can be prepared using a suitable mask. Furthermore, UV-initiated polymerization is typically much faster than thermally-initiated polymerization.
The first demonstration of a polyacrylamide monolith was performed in 1989 by Hjertén's group15. Acrylic acid and N,N′-methylenebisacrylamide were used as monomer and crosslinker, respectively, to prepare a macroporous gel plug for cation-exchange chromatography of proteins. Favorable chromatographic behavior (i.e., high efficiency at high mobile phase flow rate) was observed although the polymer monolith was compressible.
The preparation of a rigid polyacrylamide-co-bisacrylamide monolith was performed in 1997 by Svec's group19. Several variables were studied to prepare a flow-through monolith with a mean pore diameter of ˜1 μm. The porogens used for preparing the acrylamide-co-bisacrylamide monolith were dimethyl sulfoxide and a long chain alcohol, such as heptanol or dodecanol. The concentration of initiator was also investigated to adjust the medium pore diameter of the monolith; a lower concentration of initiator increased the permeability of the resulting monolith as expected. Unfortunately, thermally initiated polymerization was used to prepare the monolith. As a result, 24 h was required to complete the polymerization at 1% initiator concentration.
TABLE ACited References1.C. J. R. Morris, P. Morris, Separation Methods in Biochemistry. Wiley, New York,1976, p. 413-470.2.S. Hjerten, M. J. Zhu, J. Chromatogr. 346 (1985) 265.3.S. Hjerten, J. Chromatogr. 347 (1985) 191.4.N. J. Clarke, A. J. Tomlinson, G. Schomburg, S. Naylor, Anal. Chem. 69(1997) 2786.5.N. Iki, E. S. Yeung, J. Chromatogr A 731 (1996) 273.6.J. Preisler, E. S. Yeung, Anal. Chem. 68 (1996) 2885.7.R. McCormick, Anal. Chem. 60 (1988) 2322.8.Z. Zhao, A. Malik, M. L. Lee, Anal. Chem. 65 (1993) 2747.9.R. G. Chapman, E. Ostuni, M. N. Liang, G. Meluleni, E. Kim, L. Yan, G. Pier,H. S. Warren, G. M. Whitesides, Langmuir 17 (2001) 1225.10.E. Ostuni, R. G. Chapman, R. E., Holmlin, S. Takayama, G. M. Whitesides,Langmuir 17 (2001) 5605.11.E. Ostuni, R. G. Chapman, M. N. Liang, G. Meluleni, G. Pier, D. E. Ingber,G. M. Whitesides, Langmuir 17 (2001) 6336.12.T. Zewert, M. Harrington, Electrophoresis 13 (1992) 817.13.T. Zewert, M. Harrington, Electrophoresis 13 (1992) 824.14.J. V. Darkins, N. P. Gabbott, Polymer 22 (1981) 291.15.S. Hjertén, J. L. Liao, R. Zhang, J. Chromatogr. 473 (1989) 273.16.S. M. Fields, Anal. Chem. 68 (1996) 2709.17.H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal.Chem. 68 (1996) 3498.18.F. Svec, J. M. J. Fréchet, Anal. Chem. 54 (1992) 820.19.S, Xie, F. Svec, J. M. J, Fréchet, J. Polym. Sci. A: Polym. Chem. 35 (1997)1013.20.C. Yu, M. H. Davey, F. Svec, J. M. J. Fréchet, Anal. Chem. 73 (2001) 508821.P. H. Humble, R. T. Kelly, A. T. Woolley, H. D. Tolley, M. L. Lee, Anal.Chem. 76 (2004) 5641.22.C. Yu, M. Xu, F. Svec, J. M. J. Fréchet, J. Polym. Sci. A: Polym. Chem. 40(2002) 755.23.D. S. Peterson, T. Rohr, F. Svec, J. M. J. Fréchet, Anal. Chem. 74 (2002) 4081.24.J. J. Meyers, A. I. Liapis, J. Chromatogr. A 852 (1999) 3.25.A. I. Liapis, J. J. Meyers, O. K. Crosser, J. Chromatogr. A 865 (1999) 13.26.F. Nevejans, M. Verzele, J. Chromatogr. 350 (1985) 145.27.C. T. Mant, R. S. Hodges (Editors), High-Performance LiquidChromatography of Peptides and Proteins: Separation, Analysis, andConformation. CRC Press, Boca Raton, FL, 1991, p. 139-142.28.G. Szabo, K. Offenmuller, E. Csato, Anal. Chem. 60 (1988) 213.29.S. Lubbad, M. R. Buchmeiser, Macromol. Rapid Commun. 23 (2002) 617.30.I. Halasz, K. Martin, Angwew. Chem. (Int. Ed. Engl.) 17 (1978) 901.31.M. Al-Bokari, D. Cherrak, G. Guiochon, J. Chromatogr. A 975 (2002) 275.32.D. E. Schmidt, R. Glese, D. Conron, B. Karger, Anal. Chem. 52 (1980) 177.33.J. K. Towns, F. E. Regnier, Anal. Chem. 63 (1991) 1126.34.K. K. C. Yeung, C. A. Lucy, Anal. Chem. 69 (1997) 3435.35.J. Cunliffe, N. E. Baryla, C. A. Lucy, Anal. Chem. 74 (2002) 776.36.C. T. Culbertson, J. W. Jorgensen, Anal. Chem. 66 (1994) 955.37.D. G. McLaren, D. D. Chen, Electrophoresis, 24 (2003) 2887.38.Kimura, H.; Tanigawi, T.; Morisaka, H.; Ikegami, T.; Hosoya, K.; Ishizuka,N.; Minakuchi, H.; Nakanishi, K.; Ueda, M.; Cabrera, K.; Tanaka, N. J. Sep. Sci. 2004,27, 897-904.39.Gu, B; Armenta, J. M.; Lee, M. L. J. Chromatogr. A 2005, 1079, 382-391.40.Guyot, A.; Bartholin, M. Prog. Polym. Sci. 1982, 8, 277-332.41.Sederel, W. L.; Jong, G. J. J. Appl. Polym. Sci. 1973, 17, 2835-2846.42.Kun, K. A.; Kunin, R. J. Polym. Sci.: Part A1 1968, 6, 2689-2701.43.Svec, F. LC-GC, Europe 2003, 16(6a), 24-28.44.Svec, F. J. Sep. Sci. 2004, 27, 747-766.45.Svec, F. J. Sep. Sci. 2004, 27, 1419-1430.46.Burke, T. W. L.; Mant, C. T.; Black, J. A.; Hodges, R. S. J. Chromatogr.1989, 476, 377-389.47.Mant, C. T.; Hodges, R. S. In High-Performance Liquid Chromatographyof Peptides and Proteins: Separation, Analysis, and Conformation; Mant, C. T.;Hodges, R. S., Ed.; CRC Press: Boca Raton, 1991; pp 171-185.48.Alpert, A. J.; Andrews, P. C. J. Chromatogr. 1988, 443, 85-96.49.Imamura, T.; Sugihara, J.; Yokata, E.; Kagimoto, M.; Naito, Y.; Yanase,T. J. Chromatogr. 1984, 305, 456-460.50.Kawasaki, H.; Imajoh, S.; Suzuki, K. J. Biochem. 1987, 102, 393-400.51.Stadalius, A. A.; Quarry, M. A.; Snyder, L. R. J. Chromatogr. 1985, 327, 93-113.52.Mant, C. T.; Hodges, R. S. In High-Performance Liquid Chromatographyof Biological Macromolecules: Methods and Applications; Gooding, K.;Regnier, F., Eds.; Marcel Dekker: New York, 1990; pp 301-332.53.Mant, C. T.; Hodges, R. S. J. Chromatogr. 1985, 326, 349-356.54.Mant, C. T.; Hodges, R. S. J. Chromatogr. 1985, 327, 147-155.55.Crimmins, D. L.; Thoma, R. S.; McCourt, D. W.; Schwartz, B. D. Anal.Biochem. 1989, 176, 255-260.56.Crimmins, D. L.; Gorka, J.; Thoma, R. S.; Schwartz, B. D. J. Chromatogr.1988, 443, 63-71.57.Viklund, C.; Svec, F.; Fréchet, J. M. J. Biotechnol. Prog. 1997, 13, 597-600.58.Ueki, Y.; Umemura, T.; Li, J.; Odake, T.; Tsunoda, K. Anal. Chem. 2004,76, 7007-7012.59.Zakaria, P.; Hutchinson, J. P.; Avdalovic, N.; Liu, Y.; Haddad, P. R. Anal.Chem. 2005, 77, 417-423.60.Hilder, E. F.; Svec, F.; Fréchet, J. M. J. J. Chromatogr. A 2004, 1053,101-106.61.Righetti, P. G. in Immobilized pH Gradients. Theory and Methodology;Burdon, R. H.; van Knippenberg, P. H., Eds.; Elsevier: New York, 1990; pp 17.62.Issa, R. M.; El-Sonbati, A. Z.; El-Bindary, A. A.; Kera, H. M. J. Inorg.Organomet. Polym. 2003, 13, 269-283.63.Rivas, B.; Martinez, E.; Pereira, E.; Geckeler, K. E. Polym. Int., 2001, 50, 456-462.64.Haddad, P. R.; Jackson, P. E. Ion Chromatography: Principles andApplications; Elsevier: New York; 1990.65.Viklund, C.; Irgum, K. Macromolecules 2000, 33, 2539-2544.66.Paull, B.; Riordain, C. O.; Nesterenko, P. N. Chem. Commun. 2005, 2, 215-217.67.Guo, D.; Mant, C. T.; Taneja, A. K.; Parker, J. M. R.; Hodges, R. S. J.Chromatogr. 1986, 359, 499-517.